Shotgun Protein Sequencing: Assembly of Peptide Tandem Mass Spectra from Mixtures of Modified Proteins

peer-reviewed · Molecular & Cellular Proteomics · 2007

peer-reviewed · Molecular & Cellular Proteomics · 2007. Nuno Bandeira et al. Despite significant advances in the identification of known proteins, the analysis of unknown proteins by…
Date 2007-07-01
Type peer-reviewed
Venue Molecular & Cellular Proteomics
Publisher Elsevier BV
Contribution algorithm
DOI 10.1074/mcp.m700001-mcp200
Citations (OpenAlex) 90
Venue 2-year citedness 4.69

Abstract

Despite significant advances in the identification of known proteins, the analysis of unknown proteins by MS/MS still remains a challenging open problem. Although Klaus Biemann recognized the potential of MS/MS for sequencing of unknown proteins in the 1980s, low throughput Edman degradation followed by cloning still remains the main method to sequence unknown proteins. The automated interpretation of MS/MS spectra has been limited by a focus on individual spectra and has not capitalized on the information contained in spectra of overlapping peptides. Indeed the powerful shotgun DNA sequencing strategies have not been extended to automated protein sequencing. We demonstrate, for the first time, the feasibility of automated shotgun protein sequencing of protein mixtures by utilizing MS/MS spectra of overlapping and possibly modified peptides generated via multiple proteases of different specificities. We validate this approach by generating highly accurate de novo reconstructions of multiple regions of various proteins in western diamondback rattlesnake venom. We further argue that shotgun protein sequencing has the potential to overcome the limitations of current protein sequencing approaches and thus catalyze the otherwise impractical applications of proteomics methodologies in studies of unknown proteins.

Authors

  1. Nuno Bandeira · University of California San Diego, University of California System
  2. Karl R. Clauser · Broad Institute of the Massachusetts Institute of Technology and Harvard, Millennium Pharmaceuticals, University of California, San Francisco
  3. Pavel A. Pevzner · Max Planck Institute of Molecular Cell Biology and Genetics, Saint Petersburg Academic University, St Petersburg University, St. Petersburg State University, University of California San Diego, University of Southern California

Methods and tools

  • Shotgun Protein Sequencing: Assembles overlapping MS/MS spectra of related peptides into contigs before any sequence is called, so a protein is sequenced from the assembly rather than from individual spectra. Tolerates mixtures and unanticipated modifications.

Cites (12)

Cited by (30)

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